In this project we determined antigenic relationships based on VP4 and VP7 neutralization specificities of various rotavirus strains derived from humans and animals. The elucidation of the neutralization specificities of rotavirus is important in order to achieve a more comprehensive understanding of rotavirus epidemiology and for formulation of an effective strategy for vaccination. By using various single gene substitution human x human or human x bovine rotavirus reassortants and hyperimmune guinea pig antiserum raised against each reassortant, we successfully characterized the VP4 neutralization specificity of representative human rotavirus strains (Wa, P, WI61, DS-1, M37, ST3, PA151, K8, 59M, 69M, HCR3, Ro1845, PA169, and HAL1166) and established seven distinct VP4 (P) serotypes based on a greaterthan 20-fold antibody difference criterion that is applied to classification of VP7 serotypes. By using such reference antisera, we determined that (i) G9 US1205 strain carried P2A specificity and (ii) Malawi G8 strains MW23 and MW333 carried P2A and P1B specificity, respectively. We also showed by neutralization that Se585 strain collected in Seattle, WA, belonged to the rare G12 serotype.